Immobilized poly(n)polymerase

Abstract

The present invention relates to an immobilized poly(N)polymerase (PNP), methods of producing said PNP and uses thereof. Further disclosed is an enzyme reactor and kit comprising the PNP for producing polynucleotidylated ribonucleic acid (poly(N)RNA) molecules which are useful in gene therapy, immunotherapy, protein replacement therapy and/or vaccination.

1 . Poly(N)polymerase characterized in that the poly(N)polymerase is a bacterial poly(N)polymerase and immobilized onto a solid support. 
     
     
         2 . The poly(N)polymerase according to  claim 1 , wherein the poly(N)polymerase is selected from the group consisting of poly(A)polymerase, poly(U)polymerase, poly(G)polymerase and poly(C)polymerase. 
     
     
         3 . The poly(N)polymerase according to  claim 1  or  2 , wherein the poly(N)polymerase is a poly(A)polymerase. 
     
     
         4 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase is immobilized onto the solid support by covalent binding, affinity binding, or physical adsorption. 
     
     
         5 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase is immobilized onto the solid support by covalent binding. 
     
     
         6 . The poly(N)polymerase according to any one of the preceding claims, wherein, the poly(N)polymerase is immobilized by covalent binding to a thiol-activated solid support, haloacetyl functionalized solid support, epoxy-functionalized solid support, pyridyl disulfide-functionalized solid support, maleimide-activated solid support or a mixture thereof, preferably the poly(N)polymerase is immobilized by covalent binding to a thiol-activated solid support, haloacetyl functionalized solid support, pyridyl disulfide-functionalized solid support, maleimide-activated solid support or a mixture thereof. 
     
     
         7 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase is immobilized via a thiol group of at least one cysteine residue. 
     
     
         8 . The poly(N)polymerase according to any one of  claims 4  to  7 , wherein the covalent binding is a disulfide bridge, thioester bond or a thioether bond, preferably the covalent binding is a disulfide bridge or a thioether bond. 
     
     
         9 . The poly(N)polymerase according to any one of the preceding claims, wherein the solid support comprises a member selected from the group consisting of sepharose, thiopropyl-sepharose, sephadex, agarose, silica, magnetic beads, methacrylate beads, and nanoparticles, preferably the solid support comprises a member selected from the group consisting of sepharose, thiopropyl-sepharose, sephadex, agarose, silica, magnetic beads, and nanoparticles. 
     
     
         10 . The poly(N)polymerase according to any one of the preceding claims, wherein the solid support comprises a reactive group selected from the group consisting of thiol, haloacetyl, pyridyl disulfide, epoxy, maleimide and mixtures thereof. 
     
     
         10 . The poly(N)polymerase according to any one of the preceding claims, wherein the solid support is selected from the group consisting of activated thiol sepharose, thiopropyl-sepharose, thiol-activated sephadex, thiol-activated agarose, silica-based thiol-activated matrix, silica-based thiol-activated magnetic beads, pyridyl disulfide-functionalized nanoparticles, maleimide-activated agarose and mixtures thereof. 
     
     
         11 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase is derived from  Escherichia coli, Streptomyces coelicolor, Meiothermus silvanus, Bacillus subtilis, Thermus aquaticus, Shigella flexneri, Shigella dysenteriae, Citrobacter koseri, Salmonella bongori, Salmonella enterica, Trabulsiella guamensis, Kluyvera ascorbata, Citrobacter freundii, Enterobacter cloacae, Enterococcus gallinarum, Grimontia indica , or  Salinivibrio costicola.    
     
     
         12 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase comprises an amino acid sequence being at least 80% identical to an amino acid sequence as depicted in any one of SEQ ID NOs: 1 to 22, 24 to 155, or 203, preferably comprises an amino acid sequence being at least 80% identical to any one of SEQ ID NOs: 1, 2, 3, 16 to 22, 24 to 155, or 203, more preferably at least 80% identical to any one of SEQ ID NOs: 17 to 22, 32 to 83, 85-111, 113-139, 141-145, 146-148, 150-152, 154-155, or 203, even more preferably at least 80% identical to any one of SEQ ID NOs: 18, 58-83, 85-111, 113-139, or 203, and most preferably at least 80% identical to SEQ ID NO: 113. 
     
     
         13 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase comprises at least one newly introduced cysteine residues compared to a native poly(N)polymerase, preferably the poly(N)polymerase comprises an amino acid sequence as depicted in any one of SEQ ID NOs: 17-22, 32-155, or 203. 
     
     
         14 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase comprises only one cysteine residue or is mutated to comprise only one cysteine residue, preferably the poly(N)polymerase comprises an amino acid sequence as depicted in any one of SEQ ID NOs: 2, 16-22, 24-83, 85-111, 113-139, 141-144, 146-148, 150-152, 154-155, or 203. 
     
     
         15 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase comprises a linker element as depicted in SEQ ID Nos: 156-180. 
     
     
         16 . The poly(N)polymerase according to any one of the preceding claims, wherein the poly(N)polymerase comprises a purification tag as depicted in SEQ ID NOs: 181-201. 
     
     
         17 . Method for producing the poly(N)polymerase, preferably being a poly(A)polymerase, of any one of  claims 1  to  16 , comprising a step of
 a) contacting the poly(N)polymerase with a solid support under conditions suitable for immobilizing the poly(N)polymerase to the solid support by covalent binding, affinity binding, or physical adsorption. 
 
     
     
         18 . The method according to  claim 17 , wherein step a) comprises the formation of a disulfide bridge or thioether bond. 
     
     
         19 . The method according to  claim 17  or  18 , wherein step a) comprises the formation of a covalent bond between a cysteine residue of the poly(N)polymerase and a thiol group, a haloacetyl group, an epoxy group, a pyridyl disulfide or a maleimide group of the solid support. 
     
     
         20 . The method according to any one of  claims 17  to  19 , wherein the solid support is a thiol-activated solid support, haloacetyl functionalized solid support, pyridyl disulfide-functionalized solid support, epoxy activated solid support, or maleimide-activated solid support. 
     
     
         21 . The method according to any one of  claims 17  to  20 , further comprising prior to step a) a step of
 b) expressing the poly(N)polymerase in a suitable expression host. 
 
     
     
         22 . The method according to any one of  claims 17  to  21 , further comprising prior to step a) and, if present, after step b) a step of
 c) purifying the poly(N)polymerase from an expression host. 
 
     
     
         23 . The method according to  claim 22 , wherein step c) comprises purifying the poly(N)polymerase via affinity chromatography, preferably, the poly(N)polymerase comprises an affinity tag as depicted in any one of SEQ ID NOs: 181-201. 
     
     
         24 . The method according to any one of  claims 17  to  23 , wherein the poly(N)polymerase is a bacterial poly(N)polymerase. 
     
     
         25 . The method according to any one of  claims 17  to  24 , wherein the poly(N)polymerase is selected from the group consisting of poly(A)polymerase, poly(U)polymerase, poly(G)polymerase and poly(C)polymerase, preferably the poly(N)polymerase is a poly(A)polymerase. 
     
     
         26 . Use of a poly(N)polymerase, preferably being a poly(A)polymerase, being immobilized onto a solid support for producing polynucleotidylated ribonucleic acid (poly(N)RNA) molecules, preferably polyadenylated ribonucleic acid (poly(A)RNA) molecules. 
     
     
         27 . The use according to  claim 26 , wherein the poly(N)polymerase is the poly(N)polymerase according to any one of  claims 1  to  16  or a poly(N)polymerase obtainable by the method according to any one of  claims 17  to  25 . 
     
     
         28 . The use according to  claim 26  or  27 , comprising a step of i) contacting the poly(N)polymerase with RNA molecules and nucleotides under conditions suitable for forming a covalent bond between the nucleotides and the RNA molecules. 
     
     
         29 . The use according to any one of  claims 26  to  28 , wherein the RNA is messengerRNA (mRNA). 
     
     
         30 . The use according to any one of  claims 26  to  29 , wherein the nucleotides are selected from the group consisting of adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), nucleotide analogs and mixtures thereof. 
     
     
         31 . The use according to any one of  claims 26  to  30 , wherein step i) is performed for at least 5 min or is performed for at least 10 to 120 min or is performed for 180 minutes or longer. 
     
     
         32 . The use according to any one of  claims 26  to  31 , wherein in step i) 0.5 to 2 mol RNA are present; and/or wherein in step i) 50 to 500 mol nucleotides are present, preferably the nucleotides are ATP. 
     
     
         33 . The use according to any one of  claims 26  to  32 , further comprising a step of ii) isolating the poly(N)RNA molecules, optionally by filtration or chromatography. 
     
     
         34 . The use according to  claim 33 , wherein filtration comprises ultrafiltration and/or diafiltration. 
     
     
         35 . The use according to any one of  claims 26  to  34 , wherein the poly(N)RNA molecules are essentially homogenous. 
     
     
         36 . The use according to any one of  claims 26  to  35 , wherein at least 80% of the poly(N)RNA molecules are of the same length. 
     
     
         37 . The use according to any one of  claims 26  to  36  wherein each of the poly(N)RNA molecules comprises at least 120 nucleotidylates, preferably at least 120 adenylates. 
     
     
         38 . The use according to any one of  claims 26  to  37 , wherein the poly(N)RNA molecules are for use in gene therapy, immunotherapy, protein replacement therapy and/or vaccination. 
     
     
         39 . The use according to any one of  claims 28  to  38 , wherein the nucleotides are ATP and the poly(N)RNA molecules are poly(A)RNA molecules. 
     
     
         40 . Enzyme reactor comprising a poly(N)polymerase being immobilized onto a solid support or comprising a poly(N)polymerase according to any one of  claims 1  to  16  or comprising a poly(N)polymerase obtainable by the method according to any one of  claims 17  to  25 , preferably the poly(N)polymerase is a poly(A)polymerase. 
     
     
         41 . The enzyme reactor according to  claim 40 , further comprising
 a) at least one reaction module ( 11 ) comprising the immobilized poly(N)polymerase, and   b) one or more devices for measuring and/or adjusting at least one parameter selected from the group consisting of pH, salt concentration, magnesium concentration, phosphate concentration, temperature, pressure, flow velocity, RNA concentration and nucleotide concentration.   
     
     
         42 . The enzyme reactor according to  claim 40  or  41 , further comprising
 c) a capture module ( 13 ); 
 d) a feed module ( 14 ); and/or 
 e) an in vitro transcription (IVT) module ( 16 ). 
 
     
     
         43 . The enzyme reactor according to any one of  claims 40  to  42 , wherein the IVT module ( 16 ) and/or the feed module ( 14 ) comprises a device for adjusting and controlling the temperature ( 12 ). 
     
     
         44 . The enzyme reactor according to any one of  claims 40  to  43 , further comprising (f) at least one sensor unit ( 15 ), optionally at least one sensor unit ( 15 ) being present at the reaction module ( 11 ), the capture module ( 13 ) and/or the feed modules ( 14  and  16 ). 
     
     
         45 . The enzyme reactor according to any one of  claims 40  to  44 , wherein the reaction module ( 11 ) further comprises a filtration membrane ( 6 ), preferably being an ultrafiltration membrane, more preferably the filtration membrane ( 6 ) or the ultrafiltration membrane has a molecular weight cut-off in a range from 10 kDa to 500 MDa. 
     
     
         46 . The enzyme reactor according to any one of  claims 40  to  45 , wherein the capture module ( 13 ) comprises a resin to capture the produced poly(N)RNA molecules and to separate the produced nucleic acid molecules from other soluble components of the reaction mix. 
     
     
         47 . The enzyme reactor according to any one of  claims 40  to  46 , wherein the capture module ( 13 ) comprises a sensor unit ( 15 ). 
     
     
         48 . The enzyme reactor according to any one of  claims 40  to  47 , wherein the reaction module ( 11 ) further comprises a reflux module ( 19 ). 
     
     
         49 . The enzyme reactor according to any one of  claims 44  to  48 , wherein the at least one sensor unit comprises at least one ion-selective electrode, preferably being sensitive towards H + , Na + , K + , Mg 2+ , Ca 2+ , Cl −  and/or PO 4   3− . 
     
     
         50 . The enzyme reactor according to any one of  claims 40  to  49 , comprising more than one reaction module ( 11 ). 
     
     
         51 . Enzyme reactor according to  claim 40  or  50 , the reaction vessel comprising a thiol-activated solid support, haloacetyl functionalized solid support, pyridyl disulfide-functionalized solid support, epoxy-activated solid support, or maleimide-activated solid support. 
     
     
         52 . Enzyme reactor according to any one of  claims 40  to  51 , wherein the solid support is selected from the group consisting of activated thiol sepharose, thiopropyl-sepharose, thiol-activated sephadex, thiol-activated agarose, silica-based thiol-activated matrix, silica-based thiol-activated magnetic beads, pyridyl disulfide-functionalized nanoparticles, epoxy-methacrylate beads, and maleimide-activated agarose. 
     
     
         53 . Enzyme reactor according to any one of  claims 40  to  52  suitable for the use according to any one of  claims 26  to  39 . 
     
     
         54 . A kit comprising
 a poly(N)polymerase characterized in that the poly(N)polymerase is immobilized onto a solid support, preferably the poly(N)polymerase is the poly(N)polymerase according to any one of  claims 1  to  16  or the poly(N)polymerase obtainable by the method according to any one of  claims 17  to  25 ,   a poly(N)polymerase reaction buffer, and   nucleoside monophosphates, optionally a nucleotide mixture,   further optionally   an RNA polymerase, and optionally an RNA in vitro transcription buffer.   
     
     
         55 . The kit according to  claim 54 , wherein the poly(N)polymerase is a poly(A)polymerase.